Use of a Plasmodium vivax genetic barcode for genomic surveillance and parasite tracking in Sri Lanka.

BACKGROUND: Sri Lanka was certified as a malaria-free nation in 2016; however, imported malaria cases continue to be reported. Evidence-based information on the genetic structure/diversity of the parasite populations is useful to understand the population history, assess the trends in transmission patterns, as well as to predict threatening phenotypes that may be introduced and spread in parasite populations disrupting elimination programmes. This study used a previously developed Plasmodium vivax single nucleotide polymorphism (SNP) barcode to evaluate the population dynamics of P. vivax parasite isolates from Sri Lanka and to assess the ability of the SNP barcode for tracking the parasites to its origin. METHODS: A total of 51 P. vivax samples collected during 2005-2011, mainly from three provinces of the country, were genotyped for 40 previously identified P. vivax SNPs using a high-resolution melting (HRM), single-nucleotide barcode method. Minor allele frequencies, linkage disequilibrium, pair-wise FST values, and complexity of infection (COI) were evaluated to determine the genetic diversity. Structure analysis was carried out using STRUCTURE software (Version 2.3.4) and SNP barcode was used to identify the genetic diversity of the local parasite populations collected from different years. Principal component analysis (PCA) was used to determine the clustering according to global geographic regions. RESULTS: The proportion of multi-clone infections was significantly higher in isolates collected during an infection outbreak in year 2007. The minor allele frequencies of the SNPs changed dramatically from year to year. Significant linkage was observed in sample sub-sets from years 2005 and 2007. The majority of the isolates from 2007 consisted of at least two genetically distinct parasite strains. The overall percentage of multi-clone infections for the entire parasite sample was 39.21%. Analysis using STRUCTURE software (Version 2.3.4) revealed the high genetic diversity of the sample sub-set from year 2007. In-silico analysis of these data with those available from other global geographical regions using PCA showed distinct clustering of parasite isolates according to geography, demonstrating the usefulness of the barcode in determining an isolate to be indigenous. CONCLUSIONS: Plasmodium vivax parasite isolates collected during a disease outbreak in year 2007 were more genetically diverse compared to those collected from other years. In-silico analysis using the 40 SNP barcode is a useful tool to track the origin of an isolate of uncertain origin, especially to differentiate indigenous from imported cases. However, an extended barcode with more SNPs may be needed to distinguish highly clonal populations within the country.

Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons.

BACKGROUND: Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). METHODS: Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. RESULTS: Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. CONCLUSION: This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.